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Title: Copper binding of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase in Staphylococcus aureus
Author: Stevenson, Jack Alan
ISNI:       0000 0004 6423 6549
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2016
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Staphylococcus aureus is a Gram positive bacterium which is predominantly commensal, but which can also be a human and animal pathogen, able to cause serious infections. It is becoming an increasing problem due to it becoming resistant to modern antibiotics. In common with most bacteria, S. aureus requires small quantities of the essential metal copper, but they also experience toxicity when exposed to high concentrations of copper, a metal that has been used for its antimicrobial properties for centuries. However, the mechanism of such toxicity remains elusive. Here, the effect of copper toxicity on S. aureus has been investigated in order to understand how excess copper ions affect its physiology. The growth of S. aureus was found to be inhibited in media containing elevated copper, and the extent of this inhibition was shown to be dependent on the type of growth medium used. Analysis of soluble extracts from S. aureus cells exposed to elevated copper led to the identification of a cytoplasmic enzyme, GapA, which binds copper. GapA is a member of the wellcharacterised family of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, and is not a metalloprotein. Copper inhibition of the activity of the S. aureus GapA enzyme was demonstrated, both in S. aureus cell extracts and with recombinant enzyme in vitro, using a specific enzyme activity assay. Analysis of the purified recombinant GapA enzyme in vitro demonstrated a copper binding stoichiometry of one Cu(I) ion to each GapA monomer using analytical sizeexclusion chromatography and spectrophotometry, and evidence is presented that suggests high affinity Cu(I) binding of biochemical relevance. Some preliminary evidence is provided that the Cu(I) binding site on the GapA protein includes the thiol group of cysteine 151 located within the active site, consistent with predictions based on published crystal structures, and explaining the observed copper-dependent inhibition of enzyme activity. This is the first evidence of copper binding to a non-metalloprotein within the cytoplasm of S. aureus and adds to mounting evidence that aberrant binding of copper to cytosolic proteins contributes to bacterial copper toxicity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available